Cloning and characterization of NADP-mannitol dehydrogenase cDNA from the button mushroom, Agaricus bisporus, and its expression in response to NaCl stress.

Abstract

Mannitol, a six-carbon sugar alcohol, is the main storage carbon in the button mushroom, Agaricus bisporus. Given the physiological importance of mannitol metabolism in growth, fruit body development, and salt tolerance of A. bisporus, the enzyme responsible for mannitol biosynthesis, NADP-dependent mannitol dehydrogenase (MtDH) (EC 1.1.1.138), was purified to homogeneity, and MtDH cDNA was cloned, sequenced, and characterized. To our knowledge, this represents the first report on the isolation of a cDNA encoding an NADP-dependent mannitol dehydrogenase. The MtDH cDNA contains an open reading frame of 789 bp encoding a protein of approximately 28 kDa. The N-terminal and internal amino acid sequences of the deduced protein exactly matched the ones determined from the purified MtDH subunit, whereas the amino acid composition of the deduced protein was nearly identical to that of the purified MtDH. The MtDH cDNA showed high homology with a plant-induced short-chain dehydrogenase from Uromyces fabae. Phylogenetic analysis based on amino acid sequences from mannitol(-1-phosphate) dehydrogenases indicated a close relationship between the substrate specificity of the enzymes and phylogenetic differentiation. Salt-stressed fruit bodies showed an overall increase in mannitol biosynthesis, as was evident from the increase in MtDH activity, MtDH abundance, and MtDH RNA accumulation. Furthermore, the MtDH transcript level seems to be under developmental control, as MtDH RNA accumulated during maturation of the fruit body.

Multiple sequence alignment of the amino acid sequences of the MtDH protein from A. bisporus and a short-chain dehydrogenase with an unknown function from U. fabae. Identical residues in the two sequences are marked with asterisks, while conserved substitutions are marked with dots.

Mannitol concentration (A) and MtDH specific activity (B) during fruit body development of A. bisporus U1 grown on commercial compost with addition of 0 or 150 mM NaCl to the casing layer. Bars indicate the standard errors of the means.

Effect of NaCl on MtDH protein abundance (A) and MtDH RNA accumulation (B) in different developmental stages of A. bisporus. Mushrooms were grown on commercial compost with addition of 0 or 150 mM NaCl to the casing layer. The immunoblot of crude extracts (10 μg per lane) showed one major band of 29 kDa (A). Total RNA was extracted from each developmental stage, and relative amounts of MtDH transcript (1,000 nt) were determined by RNA blot analysis (B). The blot was hybridized to the 32P-labeled MtDH fragment. Equal RNA loading of lanes was confirmed by hybridization to a ribosomal DNA probe (rRNA).

Phylogenetic tree based on the amino acid sequences of different mannitol(-1-phosphate) dehydrogenases available in FASTA databases. The tree was composed of NAD-dependent mannitol dehydrogenases of A. graveolens (mtd-ag) and L. esculentum (mtd-le); the NAD-dependent mannitol-1-phosphate dehydrogenases of S. faecalis (mtld-sf), Streptococcus mutans (mtld-sm), Bacillus stearothermophilus (mtld-bst), B. subtilis (mtld-bsu), E. coli (mtld-ec), and M. mycoides (mtld-mm); the MtDH of A. bisporus (mtdh-ab); the short-chain dehydrogenase of U. fabae (mtdh-uf); and the NAD-dependent mannitol dehydrogenases of P. fluorescens (mtlk-pf) and R. sphaeroides (mtlk-rs). Calculated matching percentages are indicated at each branch point of the dendrogram and were generated by using DNAsis v. 2.0 (Hitachi) software. The bracket encompasses dehydrogenases with either mannose, fructose, or fructose-6-phosphate as the substrate.